KR100883992B1 - Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases - Google Patents
Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases Download PDFInfo
- Publication number
- KR100883992B1 KR100883992B1 KR1020060109551A KR20060109551A KR100883992B1 KR 100883992 B1 KR100883992 B1 KR 100883992B1 KR 1020060109551 A KR1020060109551 A KR 1020060109551A KR 20060109551 A KR20060109551 A KR 20060109551A KR 100883992 B1 KR100883992 B1 KR 100883992B1
- Authority
- KR
- South Korea
- Prior art keywords
- extract
- chopi
- prevention
- compound
- present
- Prior art date
Links
- 239000000284 extract Substances 0.000 title claims abstract description 121
- 150000001875 compounds Chemical class 0.000 title claims abstract description 86
- 239000000203 mixture Substances 0.000 title claims abstract description 30
- 230000002265 prevention Effects 0.000 title claims abstract description 22
- 208000024172 Cardiovascular disease Diseases 0.000 title abstract description 12
- 244000131415 Zanthoxylum piperitum Species 0.000 title 1
- 235000008853 Zanthoxylum piperitum Nutrition 0.000 title 1
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 29
- 239000002253 acid Substances 0.000 claims abstract description 28
- 208000031226 Hyperlipidaemia Diseases 0.000 claims abstract description 10
- 208000010125 myocardial infarction Diseases 0.000 claims abstract description 10
- 206010003210 Arteriosclerosis Diseases 0.000 claims abstract description 9
- 208000011775 arteriosclerosis disease Diseases 0.000 claims abstract description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 67
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 239000004480 active ingredient Substances 0.000 claims description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N ether Substances CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 9
- 201000001320 Atherosclerosis Diseases 0.000 claims description 7
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 5
- 235000013402 health food Nutrition 0.000 claims description 3
- 239000003208 petroleum Substances 0.000 claims description 3
- 230000001225 therapeutic effect Effects 0.000 claims description 3
- 240000007313 Tilia cordata Species 0.000 claims description 2
- 150000001335 aliphatic alkanes Chemical class 0.000 claims description 2
- HIXDQWDOVZUNNA-UHFFFAOYSA-N 2-(3,4-dimethoxyphenyl)-5-hydroxy-7-methoxychromen-4-one Chemical compound C=1C(OC)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(OC)C(OC)=C1 HIXDQWDOVZUNNA-UHFFFAOYSA-N 0.000 claims 1
- YRUMDWGUXBZEPE-UHFFFAOYSA-N cyclohexane Chemical compound C1CCCCC1.C1CCCCC1 YRUMDWGUXBZEPE-UHFFFAOYSA-N 0.000 claims 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol group Chemical group [C@@H]1(CC[C@H]2[C@@H]3CC=C4C[C@@H](O)CC[C@]4(C)[C@H]3CC[C@]12C)[C@H](C)CCCC(C)C HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 52
- 230000000694 effects Effects 0.000 abstract description 44
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 11
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000029078 coronary artery disease Diseases 0.000 abstract description 8
- 238000009825 accumulation Methods 0.000 abstract description 7
- 102000001494 Sterol O-Acyltransferase Human genes 0.000 abstract description 6
- 108010054082 Sterol O-acyltransferase Proteins 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract description 5
- 208000019622 heart disease Diseases 0.000 abstract description 3
- 238000003786 synthesis reaction Methods 0.000 abstract description 3
- 102000057234 Acyl transferases Human genes 0.000 abstract 2
- 108700016155 Acyl transferases Proteins 0.000 abstract 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 25
- 238000002360 preparation method Methods 0.000 description 22
- 235000019439 ethyl acetate Nutrition 0.000 description 21
- 235000012000 cholesterol Nutrition 0.000 description 20
- 230000036541 health Effects 0.000 description 11
- 239000000843 powder Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 210000004027 cell Anatomy 0.000 description 10
- 235000013305 food Nutrition 0.000 description 10
- 230000002401 inhibitory effect Effects 0.000 description 10
- 101100203554 Homo sapiens SOAT1 gene Proteins 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000000926 separation method Methods 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 238000010898 silica gel chromatography Methods 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 235000013361 beverage Nutrition 0.000 description 7
- -1 cholesteryl ester Chemical class 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 239000000469 ethanolic extract Substances 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 239000012046 mixed solvent Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000008280 blood Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 5
- 101000642613 Homo sapiens Sterol O-acyltransferase 2 Proteins 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 210000004204 blood vessel Anatomy 0.000 description 5
- 238000004440 column chromatography Methods 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 125000004356 hydroxy functional group Chemical group O* 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000000741 silica gel Substances 0.000 description 5
- 229910002027 silica gel Inorganic materials 0.000 description 5
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000013543 active substance Substances 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 239000012230 colorless oil Substances 0.000 description 4
- 229940125782 compound 2 Drugs 0.000 description 4
- 235000015203 fruit juice Nutrition 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 230000000704 physical effect Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 235000014347 soups Nutrition 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 231100000111 LD50 Toxicity 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 235000015278 beef Nutrition 0.000 description 3
- 150000001720 carbohydrates Chemical class 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 229940126214 compound 3 Drugs 0.000 description 3
- 235000013365 dairy product Nutrition 0.000 description 3
- 210000000497 foam cell Anatomy 0.000 description 3
- 235000003599 food sweetener Nutrition 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 210000001589 microsome Anatomy 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 235000012149 noodles Nutrition 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 230000005855 radiation Effects 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000003765 sweetening agent Substances 0.000 description 3
- 235000015192 vegetable juice Nutrition 0.000 description 3
- 239000002023 wood Substances 0.000 description 3
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 241001107116 Castanospermum australe Species 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 240000005979 Hordeum vulgare Species 0.000 description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 208000035150 Hypercholesterolemia Diseases 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 235000004347 Perilla Nutrition 0.000 description 2
- 244000124853 Perilla frutescens Species 0.000 description 2
- 108010062497 VLDL Lipoproteins Proteins 0.000 description 2
- 231100000403 acute toxicity Toxicity 0.000 description 2
- 230000007059 acute toxicity Effects 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 235000021279 black bean Nutrition 0.000 description 2
- 235000007215 black sesame Nutrition 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 235000008429 bread Nutrition 0.000 description 2
- 235000010633 broth Nutrition 0.000 description 2
- 235000021329 brown rice Nutrition 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 235000009508 confectionery Nutrition 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000013312 flour Nutrition 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000003919 heteronuclear multiple bond coherence Methods 0.000 description 2
- JQOAQUXIUNVRQW-UHFFFAOYSA-N hexane Chemical compound CCCCCC.CCCCCC JQOAQUXIUNVRQW-UHFFFAOYSA-N 0.000 description 2
- 235000015243 ice cream Nutrition 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 229940041476 lactose 100 mg Drugs 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 235000013599 spices Nutrition 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000012916 structural analysis Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- 241000701447 unidentified baculovirus Species 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 244000056139 Brassica cretica Species 0.000 description 1
- 235000003351 Brassica cretica Nutrition 0.000 description 1
- 235000003343 Brassica rupestris Nutrition 0.000 description 1
- 235000002566 Capsicum Nutrition 0.000 description 1
- 206010065559 Cerebral arteriosclerosis Diseases 0.000 description 1
- 208000018152 Cerebral disease Diseases 0.000 description 1
- 206010008111 Cerebral haemorrhage Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 240000001879 Digitalis lutea Species 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- 240000008397 Ganoderma lucidum Species 0.000 description 1
- 235000001637 Ganoderma lucidum Nutrition 0.000 description 1
- 206010060891 General symptom Diseases 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 229920001144 Hydroxy alpha sanshool Polymers 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 244000070406 Malus silvestris Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- AFCARXCZXQIEQB-UHFFFAOYSA-N N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CCNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 AFCARXCZXQIEQB-UHFFFAOYSA-N 0.000 description 1
- RJECHNNFRHZQKU-UHFFFAOYSA-N Oelsaeurecholesterylester Natural products C12CCC3(C)C(C(C)CCCC(C)C)CCC3C2CC=C2C1(C)CCC(OC(=O)CCCCCCCC=CCCCCCCCC)C2 RJECHNNFRHZQKU-UHFFFAOYSA-N 0.000 description 1
- 206010058667 Oral toxicity Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 244000243872 Pelargonium tomentosum Species 0.000 description 1
- 235000011265 Pelargonium tomentosum Nutrition 0.000 description 1
- 239000006002 Pepper Substances 0.000 description 1
- 235000016761 Piper aduncum Nutrition 0.000 description 1
- 240000003889 Piper guineense Species 0.000 description 1
- 235000017804 Piper guineense Nutrition 0.000 description 1
- 235000008184 Piper nigrum Nutrition 0.000 description 1
- PSKIOIDCXFHNJA-UHFFFAOYSA-N Sanshool Natural products CC=CC=CC=CCCC=CC=CC(=O)NC(C)C PSKIOIDCXFHNJA-UHFFFAOYSA-N 0.000 description 1
- 240000003768 Solanum lycopersicum Species 0.000 description 1
- 102100036673 Sterol O-acyltransferase 2 Human genes 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 244000098338 Triticum aestivum Species 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 238000011047 acute toxicity test Methods 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- SBXYHCVXUCYYJT-UEOYEZOQSA-N alpha-Sanshool Chemical group C\C=C\C=C\C=C/CC\C=C\C(=O)NCC(C)C SBXYHCVXUCYYJT-UEOYEZOQSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000021016 apples Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- QKSKPIVNLNLAAV-UHFFFAOYSA-N bis(2-chloroethyl) sulfide Chemical compound ClCCSCCCl QKSKPIVNLNLAAV-UHFFFAOYSA-N 0.000 description 1
- 238000009534 blood test Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 235000014121 butter Nutrition 0.000 description 1
- 235000012970 cakes Nutrition 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 210000001627 cerebral artery Anatomy 0.000 description 1
- 201000002676 cerebral atherosclerosis Diseases 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- YRUWVQGOGCKBBL-UHFFFAOYSA-N chloroform;sodium Chemical compound [Na].ClC(Cl)Cl YRUWVQGOGCKBBL-UHFFFAOYSA-N 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- RJECHNNFRHZQKU-RMUVNZEASA-N cholesteryl oleate Chemical compound C([C@@H]12)C[C@]3(C)[C@@H]([C@H](C)CCCC(C)C)CC[C@H]3[C@@H]1CC=C1[C@]2(C)CC[C@H](OC(=O)CCCCCCC\C=C/CCCCCCCC)C1 RJECHNNFRHZQKU-RMUVNZEASA-N 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 229940125773 compound 10 Drugs 0.000 description 1
- 235000014510 cooky Nutrition 0.000 description 1
- 210000004351 coronary vessel Anatomy 0.000 description 1
- 238000005100 correlation spectroscopy Methods 0.000 description 1
- 235000012495 crackers Nutrition 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000000185 dioxinlike effect Effects 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 235000013882 gravy Nutrition 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 210000003547 hepatic macrophage Anatomy 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000003987 high-resolution gas chromatography Methods 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 239000000077 insect repellent Substances 0.000 description 1
- 201000005851 intracranial arteriosclerosis Diseases 0.000 description 1
- ZLVXBBHTMQJRSX-VMGNSXQWSA-N jdtic Chemical compound C1([C@]2(C)CCN(C[C@@H]2C)C[C@H](C(C)C)NC(=O)[C@@H]2NCC3=CC(O)=CC=C3C2)=CC=CC(O)=C1 ZLVXBBHTMQJRSX-VMGNSXQWSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 210000001853 liver microsome Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 231100000668 minimum lethal dose Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 235000010460 mustard Nutrition 0.000 description 1
- 239000012454 non-polar solvent Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- XDUHQPOXLUAVEE-BPMMELMSSA-N oleoyl-CoA Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)[C@@H](O)C(=O)NCCC(=O)NCCSC(=O)CCCCCCC\C=C/CCCCCCCC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 XDUHQPOXLUAVEE-BPMMELMSSA-N 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 231100000418 oral toxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 229940124595 oriental medicine Drugs 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 235000013550 pizza Nutrition 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 230000010287 polarization Effects 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 208000020016 psychiatric disease Diseases 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 235000013580 sausages Nutrition 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 235000011888 snacks Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 239000012453 solvate Substances 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 229960002920 sorbitol Drugs 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000005507 spraying Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000012536 storage buffer Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 210000002268 wool Anatomy 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/75—Rutaceae (Rue family)
- A61K36/758—Zanthoxylum, e.g. pricklyash
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/326—Foods, ingredients or supplements having a functional effect on health having effect on cardiovascular health
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Botany (AREA)
- Vascular Medicine (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Urology & Nephrology (AREA)
- Alternative & Traditional Medicine (AREA)
- Biotechnology (AREA)
- Cardiology (AREA)
- Medical Informatics (AREA)
- Diabetes (AREA)
- Heart & Thoracic Surgery (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
본 발명은 초피 추출물 또는 이로부터 분리한 화합물을 포함하는 심장순환계 질환의 예방 및 치료용 조성물에 관한 것으로, 구체적으로 초피나무 지상부 또는 열매껍질에서 추출한 초피 추출물 및 이로부터 분리한 산스울계 화합물을 포함하는 심장질환계 질환의 예방 및 치료용 조성물에 관한 것이다. 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물은 아실-코에이:콜레스테롤 아실전이효소 (acyl-CoA:cholestyerol acyltransferase)의 활성을 효과적으로 억제하므로, 콜레스테릴 에스테르의 합성 및 축적에 의해 유발되는 고지혈증, 동맥경화, 관상동맥 심장병 및 심근경색 등과 같은 심장순환계 질환의 예방에 유용하게 사용될 수 있다.The present invention relates to a composition for the prevention and treatment of heart circulatory diseases, including Chopi extract or a compound isolated therefrom, and specifically comprising Chopi extract extracted from the ground or fruit bark of Chopi tree and Sanswool compound separated therefrom. The present invention relates to a composition for preventing and treating heart disease. Chopi extract of the present invention or the acid-based compound isolated therefrom effectively inhibits the activity of acyl-CoA: cholestyerol acyltransferase (acyl-CoA: cholestyerol acyltransferase), which is caused by the synthesis and accumulation of cholesteryl esters It can be usefully used for the prevention of cardiovascular diseases such as hyperlipidemia, arteriosclerosis, coronary heart disease and myocardial infarction.
초피 추출물, 산스울계 화합물, 아실-코에이:콜레스테롤 아실전이효소, 심혈관계 질환 Chopi Extract, Sansule Compound, Acyl-Coay: Cholesterol Acyltransferase, Cardiovascular Disease
Description
본 발명은 초피 추출물 또는 이로부터 분리한 화합물을 포함하는 심장순환계 질환의 예방 및 치료용 조성물에 관한 것으로, 더욱 상세하게는 초피나무 지상부 또는 열매껍질에서 추출한 초피 추출물 및 이로부터 분리한 산스울계 화합물을 포함하는 심장질환계 질환의 예방 및 치료용 조성물에 관한 것이다. The present invention relates to a composition for the prevention and treatment of heart circulatory diseases, including Chopi extract or a compound isolated therefrom, and more particularly, Chopi extract extracted from the ground or fruit bark of Chopi tree and Sanswool compound separated therefrom. It relates to a composition for the prevention and treatment of heart disease diseases comprising.
최근 성인병 증가와 아울러 동맥경화증 등 혈관장애질환이 많이 증가하고 있다. 동맥경화증은 동맥이 비후되고 경화되어 탄력을 잃고 약해진 것으로서, 노화와 더불어 발생하는 주요 질환 중의 하나이다. 동맥경화는 뇌동맥 또는 관상동맥에서 일어나기 쉬운데, 뇌동맥경화증의 경우에는 두통, 현기증, 정신장애를 나타내고 뇌연화증의 원인이 되며 관상동맥경화증의 경우에는 심장부에 동통과 부정맥을 일으켜 협심증, 심근경색 등의 원인이 되는 것으로 알려져 있다. 또한 이로 인해 고혈압, 심장병, 뇌일혈 등이 유발되어, 동맥경화증으로 인한 질병이 현대 사회에 있어, 특히 50~60대의 남성들에게 가장 큰 사망요인으로 부각되고 있다. Recently, with the increase of adult disease, vascular disorders such as arteriosclerosis are increasing. Atherosclerosis is an artery that thickens and hardens, loses its elasticity and weakens, and is one of the major diseases that occur with aging. Atherosclerosis is more likely to occur in the cerebral artery or coronary arteries. In the case of cerebral atherosclerosis, headache, dizziness, and mental disorders are indicated. It is known to become. In addition, this causes high blood pressure, heart disease, cerebral hemorrhage, and diseases caused by arteriosclerosis are emerging as the leading causes of death in modern society, especially among men in their 50s and 60s.
심혈관계 질환은 혈중 콜레스테롤 농도가 높으면 발병하는 것으로, 혈중 콜레스테롤 농도를 줄이기 위해서는 콜레스테롤 및 지방의 섭취를 줄이는 식이요법을 시행하거나 지질대사와 관련된 효소를 저해함으로써 콜레스테롤의 흡수를 억제해야 한다. 따라서, 심혈관계 질환의 예방을 목적으로 혈중 총콜레스테롤 농도를 낮출 수 있는 약물을 개발하기 위한 연구가 활발히 진행되어 왔다. 그 결과, 인체 내에서 콜레스테롤의 생합성을 저해하는 물질들이 다수 개발되어 상품화되었다. 그러나 이러한 약물들에 의한 부작용들이 보고되면서, 이를 개선하기 위하여 최근에는 보다 선택적으로 혈중 콜레스테롤만을 조절할 수 있는 화합물을 찾기 위한 연구가 집중적으로 행해지고 있으며, 그 중 대표적인 것이 아실-코에이:콜레스테롤 아실전이효소(acyl-CoA:cholesterol acyltransferase; ACAT) 억제제에 관한 것이다.Cardiovascular disease occurs when blood cholesterol levels are high. To reduce blood cholesterol levels, it is necessary to suppress the absorption of cholesterol by administering a diet that reduces cholesterol and fat intake or by inhibiting enzymes related to lipid metabolism. Therefore, research has been actively conducted to develop drugs that can lower the total cholesterol concentration in the blood for the purpose of preventing cardiovascular diseases. As a result, many substances that inhibit the biosynthesis of cholesterol in the human body have been developed and commercialized. However, as side effects reported by these drugs have been reported, researches to find a compound that can only selectively regulate blood cholesterol in recent years have been intensively conducted. Among them, acyl-Coei: cholesterol acyltransferase (acyl-CoA: cholesterol acyltransferase; ACAT) inhibitors.
ACAT는 일반적으로 콜레스테롤을 에스테르화하는 효소로서, 그 작용 기작은 크게 체내의 세 부위(장, 간, 및 혈관벽 세포)에서 일어난다.ACAT is an enzyme that generally esterifies cholesterol, and its mechanism of action occurs largely in three parts of the body (intestinal, liver, and vascular wall cells).
첫째, 장에서 ACAT는 섭취된 콜레스테롤을 에스테르의 형태로 바꾸어 장내로 흡수되는 것을 촉진시킨다. 둘째, 외부로부터 흡수되거나 체내에서 생합성된 콜레스테롤은 간에서 VLDL(very low-density lipoprotein)이라는 운반체 안에 축적된 후 혈관을 통해 신체 각 기관으로 공급되는데, 이때 ACAT에 의하여 콜레스테롤이 콜레스테릴 에스테르 형태로 전환됨으로써 운반체 내에 콜레스테롤 축적이 가능하 게 된다. 셋째, 동맥 혈관벽을 이루는 세포내에서 ACAT는 콜레스테롤을 그의 에스테르 형태로 전환시켜 세포내에 콜레스테롤이 축적되는 것을 촉진시키는데, 이는 동맥경화를 일으키는 직접적인 원인이 된다.First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol that is absorbed from the outside or biosynthesized in the body is accumulated in a carrier called very low-density lipoprotein (VLDL) in the liver and then supplied to each organ of the body through blood vessels. The conversion allows for the accumulation of cholesterol in the carrier. Third, in the cells that make up the arterial vascular wall, ACAT converts cholesterol into its ester form to promote the accumulation of cholesterol in the cell, which is a direct cause of atherosclerosis.
또한, ACAT 활성에 의해 거품세포가 콜레스테롤로부터 유도된 다량의 콜레스테릴 에스테르를 포함하기 때문에, 실험적, 임상적인 측면에서 대식세포와 평활근세포로부터 유도된 거품세포의 형성은 매우 중요하다. 혈관벽 내의 거품세포의 증식은 ACAT 활성 증가와 직접적으로 연관되어 있기 때문에 강력한 항동맥경화제로써 ACAT 저해제의 개발이 필요하다.In addition, since foam cells contain a large amount of cholesteryl ester derived from cholesterol by ACAT activity, the formation of foam cells derived from macrophages and smooth muscle cells is very important from an experimental and clinical aspect. Since the proliferation of foam cells in the vessel wall is directly related to the increase of ACAT activity, the development of ACAT inhibitors as a powerful anti-arteriosclerosis is necessary.
따라서, ACAT의 활성을 억제하는 약물은 첫째, 장내 콜레스테롤의 흡수를 억제하여 체내로 유입되는 콜레스테롤의 양을 감소시킬 수 있으며, 둘째, 간에서 혈관 내로 콜레스테롤이 방출되는 것을 억제하여 혈중 콜레스테롤 농도를 떨어뜨릴 수 있고, 셋째, 혈관벽 세포에 콜레스테롤이 축적되는 것을 방지하여 직접적으로 동맥경화를 예방할 수 있을 것으로 기대된다.Therefore, drugs that inhibit the activity of ACAT can firstly reduce the amount of cholesterol that enters the body by inhibiting the absorption of intestinal cholesterol, and secondly, by inhibiting the release of cholesterol into the blood vessels in the liver, thereby reducing blood cholesterol levels. Third, it is expected to prevent atherosclerosis by preventing cholesterol from accumulating in blood vessel wall cells.
지금까지 보고된 ACAT 활성 저해제는 쥐간 마이크로좀 ACAT 또는 쥐간 대식세포(J774) ACAT에 대한 활성 저해제가 있다. 인간의 ACAT는 인간 ACAT-1 및 인간 ACAT-2가 있는데, 인간 ACAT-1(50 kDa)은 성인의 간, 부신, 대식세포, 신장에서 주로 작용하며, 인간 ACAT-2(46 kDa)는 소장에서 작용한다(Rudel, L. L. et al., Curr. Opin. Lipidol 12: 121-127, 2001). 인간 ACAT 활성을 저해하는 물질은 음식으로부터 유입되는 콜레스테롤의 흡수를 억제하고, 혈관내벽에 콜레스테릴 에스 테르의 축적을 억제하는 기작을 통해 고콜레스테롤증, 콜레스테롤 결석 또는 동맥경화 예방 및 치료제의 표적이 되고 있다(Buhman, K. K. et al., Nature Medicine 6: 1341-1347, 2000).ACAT activity inhibitors reported so far are activity inhibitors for rat liver microsome ACAT or rat liver macrophage (J774) ACAT. Human ACAT includes human ACAT-1 and human ACAT-2. Human ACAT-1 (50 kDa) mainly acts on the liver, adrenal gland, macrophage and kidney of adult, and human ACAT-2 (46 kDa) is small intestine. (Rudel, LL et al., Curr. Opin. Lipidol 12: 121-127, 2001). Substances that inhibit human ACAT activity are the targets for preventing and treating hypercholesterolemia, cholesterol stones or atherosclerosis through mechanisms that inhibit the absorption of cholesterol from food and the accumulation of cholesteryl esters in the blood vessel walls. (Buhman, KK et al., Nature Medicine 6: 1341-1347, 2000).
한편, 초피(제피, 젠피 : Zanthoxylum piperitum DC)는 운향과의 낙엽관목 으로서 대개 열매껍질을 향신료와 약으로 쓰고, 씨앗이나 어린 잎, 나무, 줄기도 여러 용도로 쓴다. 초피 열매(산초)는 한방에서 해독, 구충, 진통제로 쓰이고 있으며, 주로 한국, 일본 그리고 중국에 분포하고 있다. 최근에는 후추와 겨자를 능가하는 세계 제일의 천연 향신료로 사용되고 있으며, 이를 사용한 종래기술로는 대한민국 특허 제 1993-13688호에 개시된 생약(초피나무와 분지나무의 과피)을 주재로 한 약용술 및 그의 제조방법, 대한민국 특허 제 314545호에 개시된 초피나무로부터 분리한 항균물질 및 그 분리방법, 대한민국 특허 제 314545호에 개시된 초피나무 추출물을 유효성분으로 하는 다이옥신 유사물질에 대한 길항성 조성물, 대한민국 특허 제 2004-75686호에 개시된 산초 조추출물을 유효성분으로 하는 결핵치료용약학조성물 및 건강기능성식품, 대한민국 특허 제 2004-89988호에 개시된 초피속 식물 추출물을 함유하는 항코로나바이러스 조성물 등이 있으며, 초피에서 분리, 보고된 성분으로는 산스울(sanshool)계 알리페틱 에시드 아미아드(aliphatic acid amides)(Hashimoto, K. et al., Planta Med. 67: 179-181, 2001; Hatano, T. et al., Phytochemistry 65: 2599-2604, 2004; Sugai, E. et al., Biosci. Biotechnol. Biochem. 69: 1958-1962, 2005)가 알려져 있으나, 아직까지 초피 추출 물 및 이로부터 분리한 산스울계 화합물의 ACAT과 관련된 지질 대사에 미치는 영향에 대해서는 알려진 바 없다. On the other hand, Chopi (Zepi, Genpy: Z anthoxylum piperitum DC) is a deciduous shrub with rhyme and usually uses fruit peel as spice and medicine, seeds, young leaves, trees and stems. Chopi fruit (sancho) is used as a detoxification, insect repellent, and painkiller in oriental medicine, and is mainly distributed in Korea, Japan, and China. Recently, it has been used as the world's first natural spice that surpasses pepper and mustard, and the prior art using the medicinal medicine mainly based on the herbal medicine disclosed in Korean Patent No. 1993-13688 (bark of the bark and branch trees) and its Manufacturing method, antimicrobial material isolated from the bark wood disclosed in Korean Patent No. 314545, and its separation method, antagonistic composition for dioxin-like material using the bark wood extract disclosed in Korean Patent No. 314545 as an active ingredient, Korean Patent No. 2004 -75686 tuberculosis therapeutic pharmaceutical composition and the health functional food as an active ingredient as an extract of the herbaceous acid, anti-coronavirus composition containing the herbaceous plant extract disclosed in Korean Patent No. 2004-89988 In addition, the reported component is sanshool-based aliphatic acid amides (Has himoto, K. et al., Planta Med. 67: 179-181, 2001 ; Hatano, T. et al., Phytochemistry 65: 2599-2604, 2004; Sugai, E. et al., Biosci. Biotechnol. 69: 1958-1962, 2005), but there is no known effect on ACAT-related lipid metabolism of Chopi extracts and Sanswool compounds isolated therefrom.
이에 본 발명자들은 부작용이 적은 새로운 고지혈증, 동맥경화, 관상동맥 심장병 및 심근경색 등과 같은 심장순환계 질환의 예방 및 치료제를 천연물에서 탐색하던 중, 초피 추출물 및 이로부터 분리한 산스울계 화합물의 ACAT 저해 활성이 우수하다는 것을 확인하여 본 발명을 완성하였다.Therefore, the inventors of the present invention, while searching for a natural preventive agent for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, arteriosclerosis, coronary heart disease and myocardial infarction, which have few side effects, have shown that the ACAT inhibitory activity of chopi extract and Sanswool compounds isolated therefrom It confirmed that it is excellent and completed this invention.
본 발명의 목적은 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 유효성분으로 함유하는 심장순환계 질환의 예방 및 치료용 조성물을 제공하는 것이다.It is an object of the present invention to provide a composition for the prevention and treatment of cardiovascular disease, which contains Chopi extract or Sanswool compound isolated therefrom as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 초피 추출물을 유효성분으로 함유하는 심장순환계 질환의 예방 및 치료용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for the prevention and treatment of heart circulatory disorders containing Chopi extract as an active ingredient.
또한, 본 발명은 상기 초피 추출물로부터 분리한 산스울계 화합물을 유효성분으로 함유하는 심장순환계 질환의 예방 및 치료용 조성물을 제공한다.The present invention also provides a composition for the prevention and treatment of heart circulatory diseases containing the acid-based compounds isolated from the chopi extract as an active ingredient.
또한, 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 포함하는 인간 ACAT(acyl-CoA:cholesterol acyltransferase) 활성 억제제를 제공한다.In addition, the present invention provides a human ACAT (acyl-CoA: cholesterol acyltransferase) activity inhibitor comprising a chopi extract or a sanswooul compound isolated therefrom.
아울러, 본 발명은 초피 추출물로부터 분리한 산스울계 화합물의 제조방법을 제공한다.In addition, the present invention provides a method for producing a acid-based compound separated from the extract of Chopi.
이하, 본 발명을 더욱 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 초피 추출물을 유효성분으로 함유하는 심장순환계 질환의 예방 및 치료용 조성물을 제공한다.The present invention provides a composition for the prevention and treatment of cardiovascular diseases containing Chopi extract as an active ingredient.
초피 추출물은 물 또는 유기용매를 이용하여 추출하며 구체적으로, 건조된 초피(지상부 또는 열매껍질)에 용매, 바람직하게는 물, C1 ~ C6 알코올, C5 ~ C7 알칸, 클로로포름, 에틸아세테이트, 아세톤, 에테르, 벤젠, 메틸렌클로라이드(methylene chloride), 사이클로헥산(cyclohexane), 석유에테르(petroleum ether) 또는 이들의 혼합용매, 보다 바람직하게는 물, C1 ~ C6 알코올, n-헥산, 클로로포름, 에틸아세테이트, 아세톤 또는 이들의 혼합용매, 가장 바람직하게는 에탄올 또는 아세톤을 첨가하여 추출한다.Chopi extract is extracted using water or organic solvent. Specifically, the dried Chopi (solar or fruit peel) is a solvent, preferably water, C 1 ~ C 6 alcohol, C 5 ~ C 7 alkanes, chloroform, ethyl acetate , Acetone, ether, benzene, methylene chloride, cyclohexane, petroleum ether or a mixed solvent thereof, more preferably water, C 1 to C 6 alcohol, n -hexane, chloroform , Ethyl acetate, acetone or a mixed solvent thereof, most preferably ethanol or acetone is added for extraction.
추출용매는 초피나무 지상부 및 열매껍질 건중량의 1 ~ 20 배로 하는 것이 바람직하다. 추출방법은 통상적으로 행하는 모든 추출방법이 가능하며, 바람직하게는, 40 ~ 120℃에서 끊이는 열수추출하거나, 에탄올 또는 아세톤 등을 추출용매로 사용할 경우, 상온에서 1일 ~ 5일 동안 방치하여 추출할 수 있다. 상기의 방법으로 얻어진 추출물은 여과지 등을 이용한 여과, 정제, 농축 등의 단계를 거쳐 초피 추출물로서 사용될 수 있다.The extraction solvent is preferably 1 to 20 times the dry weight of the ground bark and fruit peel. The extraction method can be any extraction method usually performed, preferably, hot water extraction at 40 ~ 120 ℃, or when using ethanol or acetone as an extraction solvent, left for 1 to 5 days at room temperature Can be extracted. The extract obtained by the above method may be used as a chopped extract through the steps of filtration, purification, concentration using a filter paper or the like.
또한, 본 발명은 상기 초피 추출물로부터 분리한 산스울계 화합물을 유효성분으로 함유하는 심장순환계 질환의 예방 및 치료용 조성물을 제공한다.The present invention also provides a composition for the prevention and treatment of heart circulatory diseases containing the acid-based compounds isolated from the chopi extract as an active ingredient.
구체적으로 상기 초피나무 지상부 추출물의 클로로포름 가용 추출물로부터 β-산스울 화합물을 분리하였다. 초피나무 지상부 추출물의 n-헥산 가용 추출물로부터는 γ-산스울 화합물을 분리 할 수 있었다. 초피 열매껍질 추출물의 클로로포름 가용 추출물로부터는 하이드록시 β-산스울 화합물을 분리하였으며, 하이드록시 β-산스울 화합물로부터 순수 활성물질인 β-산스울 아세테이트를 수득하였다. Specifically, β-sansul compound was isolated from the chloroform soluble extract of the above-ground extract of the bark tree. The γ-sansool compound could be isolated from the n -hexane soluble extract of the ground extract of the bark tree. Hydroxy β-sansul compound was isolated from the chloroform soluble extract of Chopi fruit peel extract, and β-sansul acetate, a pure active substance, was obtained from the hydroxy β-sansul compound.
본 발명의 상기 산스울계 화합물은 하기 화학식 1에 나타낸 바와 같다.The acid-based compound of the present invention is as shown in the following formula (1).
R1=또는,R 1 = or ,
R2= H, OH 또는 OAC, R 2 = H, OH or OA C ,
n= 0 또는 1. n = 0 or 1.
상기 화학식 1에서 β-산스울 화합물은 R1이 이고 R2는 H이고 n은 0이다. γ-산스울 화합물은 R1이 이고 n은 1이다. 하이드록시 β-산스울 화합물은 R1이 이고 R2는 OH이고 n은 0이다. β-산스울 아세테이트 화합물은 R1이 이고 R2는 OAC이고 n은 0이다.In Formula 1, β-sansul compound is R 1 And R 2 is H and n is 0. γ-sanswool compound has R 1 And n is 1. The hydroxy β-sansul compound is R 1 And R 2 is OH and n is 0. β-sansul acetate compound has R 1 And R 2 is OA C and n is 0.
산스울계 화합물은 약학적으로 허용되는 염의 형태로 사용될 수 있으며, 통상의 방법에 의해 제조되는 모든 염, 수화물 및 용매화물, 시판되는 시약을 사용할 수 있으며, 본 발명에서는 초피로부터 추출, 분리 및 정제하여 사용한다.The acid-based compound may be used in the form of a pharmaceutically acceptable salt, and all salts, hydrates and solvates, and commercially available reagents prepared by conventional methods may be used. use.
또한, 본 발명은 초피 추출물로부터 분리한 산스울계 화합물의 제조방법을 제공한다.The present invention also provides a method for producing a acid-based compound separated from the extract of Chopi.
구체적으로 본 발명에 따른 산스울계 화합물은 당업계에 널리 알려진 방법 또는 하기와 같은 본 발명에 따라 추출된 것, 합성된 것 또는 시판중인 것을 선택하여 사용할 수 있다. 바람직하게 상기 산스울계 화합물은 본 발명에 따른 초피 (지상부 또는 열매껍질)에서 추출될 수 있다.Specifically, the acid-based compound according to the present invention may be selected and used according to a method well known in the art or extracted according to the present invention as described below, or a commercially available one. Preferably the acid-based compound may be extracted from Chopi (ground part or fruit peel) according to the present invention.
상기 본 발명에 따른 산스울계 화합물은The acid-based compound according to the present invention
(1) 초피(지상부 또는 열매껍질)를 유기용매로 추출하여 추출물을 얻는 단계; (1) extracting chopi (ground part or fruit peel) with an organic solvent to obtain an extract;
(2) 상기에서 얻은 추출물에 물을 가하여 현탁시키고, n-헥산, 클로로포름, 에틸아세테이트를 사용하여 순차적으로 분획하여 가용 추출물을 얻는 단계; 및(2) suspending by adding water to the extract obtained above, and sequentially fractionating using n -hexane, chloroform, ethyl acetate to obtain a soluble extract; And
(3) 상기 가용 추출물을 실리카겔 크로마토그래피로 화합물을 분리하고 정제하여 산스울계 화합물을 얻는 단계를 포함하는 방법에 의해 초피(지상부 또는 열매껍질)에서 추출, 분리 및 정제될 수 있다.(3) The soluble extract may be extracted, separated and purified from Chopi (ground or fruit peel) by a method comprising the step of separating and purifying the compound by silica gel chromatography to obtain a sansule-based compound.
본 발명의 초피(지상부 또는 열매껍질) 추출물 또는 이로부터 분리한 산스울계 화합물은 인간 ACAT에 대한 강한 저해활성을 나타내었다(표 1 참조). 인간 ACAT 활성을 저해하는 물질은 음식으로부터 유입되는 콜레스테롤의 흡수를 억제하고, 혈관내벽에 콜레스테릴 에스테르의 축적을 억제하는 기작을 통해 고콜레스테롤증, 콜레스테롤 결석 또는 동맥경화 예방 및 치료제의 표적이 되고 있다(Buhman, K. K. et al., Nature Medicine 6: 1341-1347, 2000). Chopi (ground part or fruit peel) extract of the present invention or the acid-based compounds isolated therefrom showed strong inhibitory activity against human ACAT (see Table 1). Substances that inhibit human ACAT activity become targets for the prevention and treatment of hypercholesterolemia, cholesterol stones or atherosclerosis through mechanisms that inhibit the absorption of cholesterol from food and the accumulation of cholesteryl esters in the blood vessel walls. (Buhman, KK et al., Nature Medicine 6: 1341-1347, 2000).
상기의 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 마우스에 경구 투여하여 독성 실험을 수행한 결과, 경구 투여 독성시험에 의한 50% 치사량(LD50)은 적어도 1,000 ㎎/㎏ 이상인 안전한 물질로 판단된다. As a result of the oral administration of the chopi extract or the Sansool-based compound isolated therefrom to mice, 50% lethal dose (LD 50 ) by oral toxicity test was determined to be a safe substance of at least 1,000 mg / kg or more. .
따라서, 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물은 인간 ACAT 활성 저해도는 높고 안전한 물질이므로 인간 ACAT에 의해 유발되는 것으로 알려진 고지혈증, 관상동맥 심장병, 동맥경화 및 심근경색과 같은 심장순환계 질환의 예방 또는 치료에 유용하게 사용될 수 있다.Therefore, the chopi extract of the present invention or the sansool-based compound isolated therefrom is a high and safe inhibitor of human ACAT activity, and cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction, which are known to be caused by human ACAT. It can be usefully used for the prevention or treatment of.
본 발명의 상기 초피 추출물 또는 이로부터 분리한 산스울계 화합물에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주사용 제형, 환약, 캡슐, 과립 또는 정제로 제제화 할 수 있다. 더 나아가 당분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The chopi extract of the present invention or the acid-based compounds separated therefrom may further contain one or more active ingredients exhibiting the same or similar functions. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, as necessary, including antioxidants, buffers, Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물은 목적하는 방법에 따라 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 초피 추출물 또는 이로부터 분리 한 산스울계 화합물의 일일 투여량은 10 ~ 2,000 ㎎/㎏ 이며, 바람직하게는 50 ~ 500 ㎎/㎏이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The compositions of the present invention may be administered parenterally (eg, intravenously, subcutaneously, intraperitoneally or topically) or orally, depending on the desired method, and the dosage may be based on the weight, age, sex, health status, The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The daily dosage of the chopi extract according to the present invention or the sansool-based compound isolated therefrom is 10 to 2,000 mg / kg, preferably 50 to 500 mg / kg, and more preferably administered once to several times a day. .
또한, 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 유효성분으로 함유하는 심장순환계 질환 예방 및 치료용 조성물은 건강식품 형태로 제공될 수 있다. In addition, the composition for preventing and treating cardiac circulatory diseases containing the chopi extract of the present invention or the acid-based compound isolated therefrom as an active ingredient may be provided in the form of health food.
본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 식품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에는 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물은 원료에 대하여 0.01 ~ 10 중량부, 바람직하게는 0.05 ~ 1 중량부의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.When the chopi extract of the present invention or the acid-based compound separated therefrom is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be suitably determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, during the manufacture of food or beverages, the extract of Chopi or sansool compound of the present invention is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 슈크로스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물 100 ㎖당 일반적으로 약 0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 ml of the chopi extract of the present invention or the sansool-based compound isolated therefrom.
상기 외에 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등에 첨가할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육에도 초피 추출물 또는 이로부터 분리한 산스울계 화합물을 첨가할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 초피 추출물 또는 이로부터 분리한 산스울계 화합물 100 중량부 당 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the extract of Chopi or sansoul-based compounds isolated therefrom are various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, It can be added to stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition to the flesh for the production of natural fruit juices, fruit juice beverages and vegetable beverages may be added Chopi extract or the acid-based compounds separated therefrom. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the chopi extract of the present invention or the acid-based compounds separated therefrom.
또한, 초피 추출물 또는 이로부터 분리된 산스울계 화합물을 포함하는 인간 ACAT(acyl-CoA:cholesterol acyltransferase) 활성 억제제를 제공한다. In addition, it provides a human acyl-CoA: cholesterol acyltransferase (ACAT) activity inhibitor comprising Chopi extract or Sanswool compound isolated therefrom.
구제적으로 인간 ACAT에는 인간 ACAT-1 및 인간 ACAT-2가 있는데, 본 발명에 의한 초피 추출물, 또는 그로부터 분리한 산스울계 화합물은 인간 ACAT-1 및 인간 ACAT-2에 대해 각각 저해 활성이 우수함을 알 수 있었다(표 1 참조). 따라서 본 발명은 인간 ACAT 활성 억제제를 제공함으로서 심장순환계 질환의 예방 및 치료에 유용하게 사용할 수 있다.In detail, human ACAT includes human ACAT-1 and human ACAT-2. Chopi extracts according to the present invention or Sanswool compounds isolated therefrom have excellent inhibitory activity against human ACAT-1 and human ACAT-2, respectively. It was found (see Table 1). Therefore, the present invention can be usefully used for the prevention and treatment of cardiovascular diseases by providing a human ACAT activity inhibitor.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예 및 실험예를 제시한다. 그러나 하기의 실시예 및 실험예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 실시예 및 실험예에 의해 본 발명의 내용이 한정되는 것은 아니다. Hereinafter, preferred examples and experimental examples are presented to help understand the present invention. However, the following Examples and Experimental Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the Examples and Experimental Examples.
<실시예 1> 초피나무 지상부 및 열매껍질 추출물의 제조<Example 1> Preparation of the ground bark and fruit bark extract
초피나무(대한민국 경상남도 거창에서 채취) 지상부 1 kg을 건조시키고 분쇄기를 사용하여 분쇄하여 분말로 만들었다. 초피나무 분말을 에탄올 2 ℓ에 첨가하여 실온에서 3일 동안 방치시켜 추출한 후, 여과지를 이용하여 여과하여 추출액 및 고상 잔류물을 얻었다. 수득된 추출액을 감압농축하여 초피나무 지상부 에탄올 추출물인 유성물질 34 g을 얻었다.1 kg of ground vinewood (from Geochang, Gyeongsangnam-do, Korea) was dried and ground to a powder using a grinder. The bark wood powder was added to 2 L of ethanol, extracted by standing at room temperature for 3 days, and then filtered using filter paper to obtain an extract and a solid residue. The obtained extract was concentrated under reduced pressure to obtain 34 g of an oily substance which is the ethanol extract of the groundwood of the bark.
초피 열매껍질(대한민국 경상남도 거창에서 구입) 500 g을 건조시키고 분쇄기를 사용하여 분쇄하여 분말로 만들었다. 초피 열매껍질 분말을 에탄올 1 ℓ에 넣어 상온에서 3일 동안 방치시켜 추출한 후, 여과지를 이용하여 여과하여 추출액 및 고상 잔류물을 얻었다. 수득된 추출액을 감압농축하여 초피열매껍질 에탄올 추출물인 유성물질 67 g을 얻었다.500 g of Chopi fruit peel (purchased in Geochang, Gyeongsangnam-do, Korea) was dried and ground using a grinder to make a powder. Chopi fruit peel powder was added to 1 l of ethanol and left for 3 days at room temperature for extraction. Then, the resultant was filtered using filter paper to obtain an extract and a solid residue. The obtained extract was concentrated under reduced pressure to obtain 67 g of an oily substance which is the ethanol extract of the husk bark shell.
상기 초피나무 지상부 또는 열매껍질의 에탄올 추출물을 이용하여 하기 실험예 1의 방법에 따라 실험한 결과, 두 추출물 모두 ACAT 활성 억제 효과가 뛰어나 이하 실시예에서 사용하였다(표 1).Experiment using the ethanol extract of the ground bark or fruit bark according to the method of Experimental Example 1 below, both extracts were excellent in the ACAT activity inhibitory effect was used in the following examples (Table 1).
<실시예 2> 초피 추출물로부터 비극성 용매 가용 추출물의 제조Example 2 Preparation of Nonpolar Solvent Soluble Extract from Chopi Extract
상기 실시예 1에서 제조한 초피나무 지상부의 에탄올 추출물에 물 500 ㎖을 가하여 현탁시키고, n-헥산(n-hexane), 클로로포름(CHCl3) 및 에틸아세테이트(EtOAc) 순으로 분획하여, 각각 n-헥산 가용 추출물(n-hexane-soluble) 8.6 g, 클로로포름 가용 추출물(CHCl3-soluble) 1.9 g 및 에틸아세테이트 가용 추출물(EtOAc-soluble) 1.3 g을 수득하였다.The embodiment was suspended by adding a 500 cho ㎖ water in the ethanol extract of the aerial part linden, prepared in example 1, n - hexane (n -hexane), chloroform (CHCl 3) and ethyl acetate (EtOAc) in the order of fractions, each of n - 8.6 g of hexane soluble extract ( n -hexane-soluble), 1.9 g of chloroform soluble extract (CHCl 3 -soluble) and 1.3 g of ethyl acetate soluble extract (EtOAc-soluble) were obtained.
상기 실시예 1에서 제조한 초피 열매껍질의 에탄올 추출물에 물 1 ℓ을 가하여 현탁시키고, n-헥산(n-hexane), 클로로포름(CHCl3) 및 에틸아세테이트(EtOAc) 순으로 분획하여, 각각 -헥산 가용 추출물(n-hexane-soluble) 19 g, 클로로포름 가용 추출물(CHCl3-soluble) 10 g 및 에틸아세테이트 가용 추출물(EtOAc-soluble) 4 g을 수득하였다.1 liter of water was added to the ethanol extract of the fruit bark of Chopi fruit prepared in Example 1, suspended, n -hexane ( n -hexane), chloroform (CHCl 3 ) and ethyl acetate (EtOAc) in the order of each, -hexane 19 g of soluble extract (n-hexane-soluble), 10 g of chloroform soluble extract (CHCl 3 -soluble) and 4 g of ethyl acetate soluble extract (EtOAc-soluble) were obtained.
상기 각각의 가용 추출물에 대해 실험예 1과 같은 방법으로 실험한 결과, 초피 지상부의 헥산 가용 추출물 및 클로로포름 가용 추출물과 초피 열매껍질의 클로 로포름 가용 추출물에서 ACAT 활성 억제 효과가 우수함을 확인하고, 하기 실시예 3, 4 또는 5에서 사용하였다(표 1).As a result of the experiment in the same manner as in Experiment 1 for each of the soluble extracts, it was confirmed that the inhibitory effect of ACAT activity is excellent in the hexane soluble extract and chloroform soluble extract and chloroform soluble extract of the fruit skin peel of Chopi ground. Used in Examples 3, 4 or 5 (Table 1).
<실시예 3> 초피나무 지상부 추출물의 클로로포름 가용 추출물로부터 <Example 3> From the chloroform soluble extract of the extract of groundwood tree ββ -산스울 화합물의 분리Separation of Sansoul Compound
상기 실시예 2에서 수득한 초피나무 지상부 추출물의 클로로포름 가용 추출물 1.9 g을 컬럼 크로마토그래피를 사용하여 분리하였다.1.9 g of the chloroform soluble extract of the groundwood extract of Example 1 from the above-derived bark tree were separated using column chromatography.
첫 번째로 실리카겔에 클로로포름 가용 추출물의 갈색 고체 물질을 흡착시킨 후, 이동상으로 n-헥산 및 에틸아세테이트(1:1, 2:1, 0:100 부피비)의 혼합용매를 이용하여 극성을 높여 가면서 실리카겔 크로마토그래피(φ50 × 150 ㎜)를 수행하여 3개(F1 ~ F3)의 분획을 얻었다. 3개 각 분획에 대해 실험예 1과 같은 방법으로 ACAT 활성 억제를 측정한 결과, 분획 1(F1)의 효과가 가장 우수하게 나타나 이를 하기 분리 과정에 사용하였다.Firstly, brown solid substance of chloroform soluble extract is adsorbed on silica gel, and then silica gel is increased by increasing polarity by using mixed solvent of n- hexane and ethyl acetate (1: 1, 2: 1, 0: 100 by volume) as mobile phase. Chromatography (φ50 × 150 mm) was performed to obtain three fractions (F1 to F3). As a result of measuring the inhibition of ACAT activity in the same manner as in Experimental Example 1 for each of the three fractions, the effect of fraction 1 (F1) was most excellent and was used in the following separation process.
ACAT 활성 억제능이 우수한 분획 1(F1, 310 ㎎)에 대해 이동상으로 n-헥산 및 에틸아세테이트(5:1 부피비)의 혼합용매를 이용하여 2차 실리카겔 크로마토그래피(φ50 × 150 ㎜)를 수행하여 8개(F1-1 ~ F1-8)의 분획을 얻었다. 8개 각 분획에 대해 실험예 1과 같은 방법으로 ACAT 활성 억제를 측정한 결과, 여섯 번째 분획(F1-6)의 효과가 가장 우수하게 나타나 이를 하기 분리 과정에 사용하였다.Fraction 1 (F1, 310 mg) having excellent ACAT activity inhibitory activity was subjected to secondary silica gel chromatography (φ50 × 150 mm) using a mixed solvent of n- hexane and ethyl acetate (5: 1 by volume) as a mobile phase. Fractions of dogs (F1-1 to F1-8) were obtained. As a result of measuring the inhibition of ACAT activity in the same manner as in Experimental Example 1 for each of the eight fractions, the effect of the sixth fraction (F1-6) was the best and was used in the following separation process.
ACAT 활성 억제능이 우수한 분획 1-6(F1-6, 77 mg)에 대해 이동상으로 100% 메탄올을 이용하여 세파덱스 LH-20 칼럼 크로마토그래피(φ1.5 × 50 ㎜)를 실시하 여 38 mg의 활성분획을 얻었다.Sepadex LH-20 column chromatography (φ1.5 × 50 mm) was carried out using 100% methanol as a mobile phase for fraction 1-6 (F1-6, 77 mg), which exhibits excellent ACAT activity. An active fraction was obtained.
이 활성분획에 대해 다시 이동상으로 n-헥산 및 에틸 아세테이트(2:1 부피비)의 혼합용액을 이용하여 실리카겔 칼럼 크로마토그래피(φ3.0 × 5.0 cm)를 실시하여 활성물질인 β-산스울 29 mg를 얻었다.This active fraction was subjected to silica gel column chromatography (φ3.0 × 5.0 cm) using a mixed solution of n -hexane and ethyl acetate (2: 1 volume ratio) again as a mobile phase, and 29 mg of β -sanswool as an active substance. Got.
<실시예 4> 초피나무 지상부 추출물의 <Example 4> Extract of the ground bark extract nn -헥산 가용 추출물로부터 From hexane soluble extract γγ -산스울 화합물의 분리Separation of Sansoul Compound
상기 실시예 2에서 수득한 초피나무 지상부 추출물의 n-헥산 가용 추출물 8.6 g을 컬럼 크로마토그래피를 사용하여 분리하였다.8.6 g of the n -hexane soluble extract of the groundwood extract of Example 1 from the above-derived bark tree was separated using column chromatography.
첫 번째로 실리카겔에 클로로포름 가용 추출물의 갈색 고체 물질을 흡착시킨 후, 이동상으로 n-헥산 및 에틸아세테이트(2:1, 1:1 부피비)과 에텔아세테이트 및 메탄올(1:1 부피비)의 혼합용매를 이용하여 실리카겔 크로마토그래피(φ50 × 150 ㎜)를 수행하여 3개 (F1 ~ F3)의 분획을 얻었다. 3개 각 분획에 대해 실험예 1과 같은 방법으로 ACAT 활성 억제를 측정한 결과, 분획 2(F2)의 효과가 가장 우수하게 나타나 이를 하기 분리 과정에 사용하였다.First, after adsorbing the brown solid material of the chloroform soluble extract on the silica gel, a mixed solvent of n -hexane and ethyl acetate (2: 1, 1: 1 volume ratio), etheracetate and methanol (1: 1 volume ratio) was used as a mobile phase. Silica gel chromatography (φ50 × 150 mm) was used to obtain three fractions (F1 to F3). As a result of measuring the inhibition of ACAT activity in the same manner as in Experimental Example 1 for each of the three fractions, the effect of fraction 2 (F2) was the best and was used in the following separation process.
ACAT 활성 억제능이 우수한 분획 2(F2, 1.4 g)에 대해 이동상으로 n-헥산 및 에틸아세테이트(5:1 부피비)의 혼합용매를 이용하여 2차 실리카겔 크로마토그래피 (φ50 × 150 ㎜)를 수행하여 5개(F2-1 ~ F2-5)의 분획을 얻었다. 5개 각 분획에 대해 실험예 1과 같은 방법으로 ACAT 활성 억제를 측정한 결과, 두 번째 분획(F2-2)의 효과가 가장 우수하게 나타나 이를 하기 분리 과정에 사용하였다.Fraction 2 (F2, 1.4 g) having excellent ability to inhibit ACAT activity was subjected to secondary silica gel chromatography (φ50 × 150 mm) using a mixed solvent of n -hexane and ethyl acetate (5: 1 by volume) as a mobile phase. Fractions of dogs (F2-1 to F2-5) were obtained. As a result of measuring the inhibition of ACAT activity in the same manner as in Experimental Example 1 for each of the five fractions, the effect of the second fraction (F2-2) was most excellent and used in the following separation process.
ACAT 활성 억제능이 우수한 분획 2-2(F2-2 76 mg)에 대해 이동상으로 100% 메탄올을 이용하여 세파덱스 LH-20 칼럼 크로마토그래피(φ1.5 × 50 ㎜)를 실시하여 39 mg의 활성분획을 얻었다.Sepadex LH-20 column chromatography (φ1.5 × 50 mm) using 100% methanol as a mobile phase was performed on fraction 2-2 (76 mg, F2-2) having excellent ability to inhibit ACAT activity. Got.
이 활성분획에 대해 다시 이동상으로 n-헥산 및 에틸 아세테이트(5:1 부피비)의 혼합용액을 이용하여 실리카겔 칼럼 크로마토그래피(φ3.0 × 5.0 cm)를 실시하여 활성물질인 γ-산스울 39 mg를 얻었다.This active fraction was subjected to silica gel column chromatography (φ3.0 × 5.0 cm) using a mixed solution of n -hexane and ethyl acetate (5: 1 volume ratio) again as a mobile phase, and 39 mg of γ -sanswool as an active substance. Got.
<실시예 5> 초피 열매껍질 추출물의 클로로포름 가용 추출물로부터 하이드록시 Example 5 Hydroxy from Chloroform Soluble Extract of Chopi Fruit Peel Extract ββ -산스울 화합물의 분리Separation of Sansoul Compound
상기 실시예 2에서 수득한 초피 열매껍질 추출물의 클로로포름 가용 추출물 10 g을 컬럼 크로마토그래피를 사용하여 분리하였다.10 g of the chloroform soluble extract of Chopi fruit peel extract obtained in Example 2 was separated using column chromatography.
첫 번째로 실리카겔에 클로로포름 가용 추출물의 갈색 고체 물질을 흡착시킨 후, 이동상으로 n-헥산 및 에틸아세테이트(1:1, 1:5, 0:100 부피비)의 혼합용매를 이용하여 극성을 높여 가면서 실리카겔 크로마토그래피(φ50 × 150 ㎜)를 수행하여 3개(F1 ~ F3)의 분획을 얻었다. 3개 각 분획에 대해 실험예 1과 같은 방법으로 ACAT 활성 억제를 측정한 결과, 분획 3(F3)의 효과가 가장 우수하게 나타나 이를 하기 분리 과정에 사용하였다.First, the brown solid substance of the chloroform soluble extract was adsorbed onto the silica gel, and then the silica gel was increased in polarity using a mixed solvent of n -hexane and ethyl acetate (1: 1, 1: 5, 0: 100 by volume) as a mobile phase. Chromatography (φ50 × 150 mm) was performed to obtain three fractions (F1 to F3). As a result of measuring the inhibition of ACAT activity in the same manner as in Experimental Example 1 for each of the three fractions, the effect of fraction 3 (F3) was the best and was used in the following separation process.
ACAT 활성 억제능이 우수한 분획 3(F3, 2.1 g)에 대해 이동상으로 n-헥산 및 에틸아세테이트(1:1 부피비)의 혼합용매를 이용하여 2차 실리카겔 크로마토그래피(φ50 × 100 ㎜)를 수행하여 활성물질인 하이드록시 β-산스울 1.3 g를 얻었다.Activated by silica gel chromatography (φ50 × 100 mm) using fractionation solvent of n -hexane and ethyl acetate (1: 1 volume ratio) as a mobile phase for fraction 3 (F3, 2.1 g) having excellent ACAT activity. 1.3 g of hydroxy β -acid wool as a substance were obtained.
<실시예 6> <Example 6> ββ -산스울 아세테이트 화합물의 제조Preparation of Sansool Acetate Compound
하이드록시 β-산스울 화합물 10 mg을 아세틱 안하이드라이드(Acetic anhydride) 2 ㎖에 녹이고 촉매량의 p-톨루엔 슬폰산(p-TsOH)을 가하였다. 반응 혼합물을 60℃에서 1 시간동안 교반한 후, 물 20 ㎖을 천천히 첨가하였다. 위 혼합물에 에틸아세테이트 30 ㎖를 가하고 2회 추출 후 황산마그네슘을 가하여 수분을 제거하였다. 여액을 감압농축하고 이동상으로 n-헥산과 에틸 아세테이트(2:1 부피비) 혼합용액을 사용하여 실리카겔 칼럼 크로마토그래피(φ20 × 100 mm)를 수행하였다. 이때 상기 목적 화합물인 순수 활성물질인 β-산스울 아세테이트 6.2 mg(수득율 54%)을 얻었다.10 mg of hydroxy β -sansul compound was dissolved in 2 ml of acetic anhydride and a catalytic amount of p -toluene sulfonic acid ( p- TsOH) was added. The reaction mixture was stirred at 60 ° C. for 1 h and then 20 ml of water was slowly added. 30 ml of ethyl acetate was added to the mixture, followed by two extractions, and magnesium sulfate was added thereto to remove moisture. The filtrate was concentrated under reduced pressure and silica gel column chromatography (φ20 × 100 mm) was performed using a mixed solution of n -hexane and ethyl acetate (2: 1 by volume) as a mobile phase. At this time, 6.2 mg (yield 54%) of β -sansul acetate which was a pure active substance of the target compound was obtained.
<실시예 7> 본 발명의 화합물의 구조 분석Example 7 Structural Analysis of Compounds of the Invention
상기 실시예 3 ~ 6을 통하여 얻은 화합물들의 구조 분석을 위하여, 하기와 같은 분석을 실시하였다. For the structural analysis of the compounds obtained in Examples 3 to 6, the following analysis was performed.
VG 고분해능 GC/MS 분광기(VG high resolution GC/MS spectrometer, HREI MS, JMS-700)를 사용하여 분자량 및 분자식을 결정하였다. 또한 핵자기 공명(NMR) 분석(Varian Unity 300 spectrometer)을 통하여 1H NMR, 13C NMR, 호모-코지(HOMO-COSY), HMQC(1 H-Detected heteronuclear Multiple-Quantum Coherence), HMBC(Heteronuclear Multiple-Bond Coherence), DEPT(Distortionless Enhancement by Polarization) 스펙트럼을 얻고, 분자구조를 결정하였다. 측정 결과는 하기와 같으며, 발표된 문헌의 것과 비교 분석한 결과, 화합물 1은 β-산스울임을 확인하였고, 화합물 3은 하이드록시-β-산스울(Yasuda I., Takeya K., Itokawa H., Pyhtochemistry, 21: 1295?1298, 1982)로 확인하였다. 화합물 2는 γ-산스울(Yasuda I., Takeya K., Itokawa H., Chem. Pharm. Bull., 29: 1791?1793, 1981)로 확인하였다.Molecular weight and molecular formula were determined using a VG high resolution GC / MS spectrometer (HRG MS, JMS-700). In addition, 1 H NMR, 13 C NMR, HOMO-COSY, HMQC ( 1 H -Detected heteronuclear M ultiple- Q uantum C oherence), HMBC through nuclear magnetic resonance (NMR) analysis (Varian Unity 300 spectrometer) (Heteronuclear Multiple-Bond Coherence), DPT (Distortionless Enhancement by Polarization) spectra were obtained, and molecular structure was determined. The measurement results are as follows. As a result of the comparative analysis, it was confirmed that the compound 1 was β -sansul, and the compound 3 was hydroxy- β -sansul (Yasuda I., Takeya K., Itokawa H. , Pyhtochemistry , 21: 1295-1298 , 1982). Compound 2 was identified as γ -sansule (Yasuda I., Takeya K., Itokawa H., Chem. Pharm. Bull. , 29: 1791-1793, 1981).
[화합물 1] [Compound 1] ββ -- 산스울Sanswool
1) 물성: 무색 오일1) Physical property: colorless oil
2) 분자량: 2472) Molecular Weight: 247
3) 분자식: C16H25NO3) Molecular Formula: C 16 H 25 NO
4) 1H-NMR (CDCl3, 300 MHz): δ 6.80 (1H, dt, J = 11.4, 5.1 Hz, H-3), 6.32 (1H, dd, J = 8.1, 5.4 Hz, H-7), 6.17-5.98 (3H, m), 5.81-5.59 (3H, m), 5.36 (1H, dt, J = 8.1, 5.4 Hz, H-6), 3.13 (t like, J = 4.8 Hz, H-1'), 2.36-2.23 (4H, m), 1.80-1.73 (4H, m), 0.91 (6H, d, J = 5.1 Hz, H-3') ; 13C-NMR (CDCl3, 300 MHz) : δ 165.8 (C-1), 143.2 (C-3), 133.3 (C-9), 131.7 (C-10), 130.0 (C-11), 129.5 (C-7), 129.4 (C-6), 125.2 (C-8), 124.1 (C-2), 40.9 (C-1'), 32.1 (C-4), 28.6 (C-2'), 26.6 (C-5), 20.2 (C-3'), 18.4 (C-12). 4) 1 H-NMR (CDCl 3 , 300 MHz): δ 6.80 (1H, dt, J = 11.4, 5.1 Hz, H-3), 6.32 (1H, dd, J = 8.1, 5.4 Hz, H-7) , 6.17-5.98 (3H, m), 5.81-5.59 (3H, m), 5.36 (1H, dt, J = 8.1, 5.4 Hz, H-6), 3.13 (t like, J = 4.8 Hz, H-1 '), 2.36-2.23 (4H, m), 1.80-1.73 (4H, m), 0.91 (6H, d, J = 5.1 Hz, H-3'); 13 C-NMR (CDCl 3 , 300 MHz): δ 165.8 (C-1), 143.2 (C-3), 133.3 (C-9), 131.7 (C-10), 130.0 (C-11), 129.5 ( C-7), 129.4 (C-6), 125.2 (C-8), 124.1 (C-2), 40.9 (C-1 '), 32.1 (C-4), 28.6 (C-2'), 26.6 (C-5), 20.2 (C-3 '), 18.4 (C-12).
5) HREIMS m/z : 247.1936 (calcd. for C16H25NO [M]+: 247.1936). 5) HREIMS m / z : 247.1936 (calcd. For C 16 H 25 NO [M] < + >: 247.1936).
[화합물 2] [Compound 2] γγ -산스울Sunwool
1) 물성: 무색 오일1) Physical property: colorless oil
2) 분자량: 2732) Molecular Weight: 273
3) 분자식: C18H27NO3) Molecular Formula: C 18 H 27 NO
4) 1H-NMR (CDCl3, 300 MHz): δ 7.18 (1H, dd, J = 15.3, 10.2 Hz, H-3), 6.33-5.97 (6H, m), 5.79-5.66 (2H, m), 5.53 (1H, s, NH), 5.36 (1H, dt, J = 10.2, 7.5 Hz, H-8), 3.16 (2H, t like, J = 6.3 Hz, H-1'), 2.35-2.23 (4H, m), 1.84-1.753 (4H, m), 0.92 (6H, d, J = 6.9 Hz, H-3'); 13C-NMR (CDCl3, 300 MHz): δ 165.8 (C-1), 141.8 (C-5), 141.0 (C-3), 133.3 (C-11), 131.8 (C-12), 130.0 (C-13), 129.9 (C-8), 129.4 (C-9), 128.7 (C-4), 125.3 (C-10), 122.2 (C-2) 46.9 (C-1'), 32.1 (C-6), 28.6 (C-2'), 27.6 (C-7), 20.0 (C-3'), 18.3 (C-14). 4) 1 H-NMR (CDCl 3 , 300 MHz): δ 7.18 (1H, dd, J = 15.3, 10.2 Hz, H-3), 6.33-5.97 (6H, m), 5.79-5.66 (2H, m) , 5.53 (1H, s, NH), 5.36 (1H, dt, J = 10.2, 7.5 Hz, H-8), 3.16 (2H, t like, J = 6.3 Hz, H-1 '), 2.35-2.23 ( 4H, m), 1.84-1.753 (4H, m), 0.92 (6H, d, J = 6.9 Hz, H-3 '); 13 C-NMR (CDCl 3 , 300 MHz): δ 165.8 (C-1), 141.8 (C-5), 141.0 (C-3), 133.3 (C-11), 131.8 (C-12), 130.0 ( C-13), 129.9 (C-8), 129.4 (C-9), 128.7 (C-4), 125.3 (C-10), 122.2 (C-2) 46.9 (C-1 '), 32.1 (C -6), 28.6 (C-2 '), 27.6 (C-7), 20.0 (C-3'), 18.3 (C-14).
5) HREIMS m/z : 273.2093 (calcd. for C18H27NO [M]+: 273.2093).5) HREIMS m / z : 273.2093 (calcd. For C 18 H 27 NO [M] < + >: 273.2093).
[화합물 3] 하이드록시 -β -산스울 [ Compound 3] hydroxy- β -sanswool
1) 물성: 무색 오일1) Physical property: colorless oil
2) 분자량: 2632) Molecular Weight: 263
3) 분자식: C16H25NO2 3) Molecular Formula: C 16 H 25 NO 2
4) 1H-NMR (CDCl3, 300 MHz): δ 6.85 (1H, dt, J = 15.3, 6.9 Hz, H-3), 6.32 (1H, m), 6.21-5.97 (4H, m), 5.87-5.66 (2H, m), 5.35 (1H, dt, J = 10.2, 7.5 Hz, H-6), 3.31 (2H, d, J = 6 Hz, H-1'), 2.37-2.23 (4H, m), 1.77 (3H, d, J = 6.6, H-12), 1.22 (6H, s, H-3'); 13C-NMR (CDCl3, 300 MHz): δ 167.2 (C-1), 144.3 (C-3), 133.6 (C-9), 131.7 (C-10), 130.2 (C-11), 129.9 (C-7), 129.5 (C-6), 125.3 (C-8), 123.9 (C-2), 50.6 (C-1'), 32.2 (C-4), 70.6 (C-2'), 26.6 (C-5), 27.2 (C-3'), 18.4 (C-12). 4) 1 H-NMR (CDCl 3 , 300 MHz): δ 6.85 (1H, dt, J = 15.3, 6.9 Hz, H-3), 6.32 (1H, m), 6.21-5.97 (4H, m), 5.87 -5.66 (2H, m), 5.35 (1H, dt, J = 10.2, 7.5 Hz, H-6), 3.31 (2H, d, J = 6 Hz, H-1 '), 2.37-2.23 (4H, m ), 1.77 (3H, d, J = 6.6, H-12), 1.22 (6H, s, H-3 '); 13 C-NMR (CDCl 3 , 300 MHz): δ 167.2 (C-1), 144.3 (C-3), 133.6 (C-9), 131.7 (C-10), 130.2 (C-11), 129.9 ( C-7), 129.5 (C-6), 125.3 (C-8), 123.9 (C-2), 50.6 (C-1 '), 32.2 (C-4), 70.6 (C-2'), 26.6 (C-5), 27.2 (C-3 '), 18.4 (C-12).
5) HREIMS m/z : 263.1885 (calcd. for C16H25NO2 [M]+: 263.1885.5) HREIMS m / z : 263.1885 (calcd. For C 16 H 25 NO 2 [M] < + >: 263.1885.
[화합물 4] β -산스울 아세테이트 [ Compound 4] β -San Wool Acetate
1) 물성: 무색 오일1) Physical property: colorless oil
2) 분자량: 3052) Molecular Weight: 305
3) 분자식: C18H27NO3 3) Molecular Formula: C 18 H 27 NO 3
4) 1H-NMR (CDCl3, 300 MHz): δ 6.85 (1H, dt, J = 15.3, 6.9 Hz, H-3), 6.32 (1H, m), 6.21-5.97 (4H, m), 5.87-5.66 (2H, m), 5.35 (1H, dt, J = 10.2, 7.5 Hz, H-6), 3.31 (2H, d, J = 6 Hz, H-1'), 2.37-2.23 (4H, m), 1.77 (3H, d, J = 6.6, H-12), 2.07 (3H, s, CH3CO-), 1.44 (6H, s, H-3'); 13C-NMR (CDCl3, 300 MHz): δ 171.9 (COCH3), 167.2 (C-1), 144.3 (C-3), 133.6 (C-9), 131.7 (C-10), 130.2 (C-11), 129.9 (C-7), 129.5 (C-6), 125.3 (C-8), 123.9 (C-2), 50.6 (C-1'), 32.2 (C-4), 70.6 (C-2'), 26.6 (C-5), 27.2 (C-3'), 21.6 (CH3CO), 18.4 (C-12). 4) 1 H-NMR (CDCl 3 , 300 MHz): δ 6.85 (1H, dt, J = 15.3, 6.9 Hz, H-3), 6.32 (1H, m), 6.21-5.97 (4H, m), 5.87 -5.66 (2H, m), 5.35 (1H, dt, J = 10.2, 7.5 Hz, H-6), 3.31 (2H, d, J = 6 Hz, H-1 '), 2.37-2.23 (4H, m ), 1.77 (3H, d, J = 6.6, H-12), 2.07 (3H, s, CH 3 CO-), 1.44 (6H, s, H-3 '); 13 C-NMR (CDCl 3 , 300 MHz): δ 171.9 (COCH 3 ), 167.2 (C-1), 144.3 (C-3), 133.6 (C-9), 131.7 (C-10), 130.2 (C -11), 129.9 (C-7), 129.5 (C-6), 125.3 (C-8), 123.9 (C-2), 50.6 (C-1 '), 32.2 (C-4), 70.6 (C -2 '), 26.6 (C-5), 27.2 (C-3'), 21.6 (CH 3 CO), 18.4 (C-12).
<실험예 1> 본 발명에 따른 초피 추출물 및 산스울계 화합물의 ACAT 활성에 미치는 영향 측정Experimental Example 1 Determination of the Effect on the ACAT Activity of Chopi Extract and Sansool Compounds According to the Present Invention
본 발명에 따른 초피 추출물, 그로부터 분리한 산스울계 화합물의 ACAT 활성에 미치는 영향을 알아보기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the effect on the ACAT activity of chopi extract according to the present invention, the acid-based compounds isolated therefrom, the following experiment was performed.
<실험예 1-1> ACAT 효소원의 제조Experimental Example 1-1 Preparation of ACAT Enzyme Source
사람 ACAT-1 및 ACAT-2의 활성에 미치는 영향을 알아보기 위하여, 베큘로바이러스 발현체제를 이용하여 hACAT-1 및 hACAT-2 각각의 단백질을 얻었다.In order to determine the effect on the activity of human ACAT-1 and ACAT-2, the proteins of hACAT-1 and hACAT-2 were obtained using a baculovirus expression system.
사람 간 cDNA 라이브러리(clontech) 스크리닝(library screening)을 통하여 얻어진 hACAT-1 및 hACAT-2 각각의 cDNA를 베큘로바이러스 전달 벡터에 삽입하고, 곤충세포인 sf9 세포에 도입하여 바이러스를 제조하였다. 다음으로, 플라크 정제(plaque purification) 방법으로 hACAT-1 및 hACAT-2 각각의 재조합 바이러스를 분리한 후, 3 차례의 증폭과정을 거쳐 저장용 바이러스(viral stock)의 역가(titer)를 높였다. 단백질 발현 효율이 좋은 Hi5 곤충세포에 재조합 바이러스를 감염다중도(Mutiplicity of Infection)가 1이 되도록 감염시킨 후, 27℃에서 하루 동안 진탕배양하였다. 배양된 hACAT-1과 hACAT-2가 각각 과량 발현된 Hi5 곤충세포들로부터 마이크로좀 분획을 분리하기 위하여, 500 × g에서 15 분간 원심분리하여 세포들을 모으고 저장 완충액(hypotonic buffer)에서 급냉동 및 급해동 방법으로 세포를 용해시킨 후, 100,000 × g에서 한 시간 동안 초원심분리하였다. 얻어진 마이크로좀 분획들은 단백질 농도가 8 ㎎/㎖이 되도록 저장완충액으로 현탁하여 사용 전까지 저온냉동기에 보관하였다.Viruses were prepared by inserting cDNAs of hACAT-1 and hACAT-2, respectively, obtained through human liver cDNA library (clontech) library screening into baculovirus delivery vectors and introducing them into sf9 cells, which are insect cells. Next, after separating the recombinant viruses of each of the hACAT-1 and hACAT-2 by plaque purification method, the titer of the storage virus (viral stock) was increased through three amplification processes. Recombinant virus was infected with Hi5 insect cells having good protein expression efficiency so as to have a multiplicity of infection (Mutiplicity of Infection) of 1, followed by shaking culture at 27 ° C for one day. To isolate the microsome fraction from Hi5 insect cells overexpressed in cultured hACAT-1 and hACAT-2, cells were collected by centrifugation at 500 x g for 15 minutes and quenched and quenched in hypotonic buffer. Cells were lysed by thawing method and then ultracentrifuged at 100,000 × g for 1 hour. The obtained microsome fractions were suspended in storage buffer so that the protein concentration was 8 mg / ml and stored in a cryocooler until use.
<실험예 1-2> ACAT 활성 측정Experimental Example 1-2 ACAT Activity Measurement
ACAT 활성의 측정은 [1-14C] 올레오일-코에이(oleoyl-CoA)(56.0 μCi/μmol; Amersham)를 기질로 하여 브레쳐 & 찬(Brecher & Chan)의 방법(Brecher, P. and Chan, C., Biochim. Biophys. Acta, 617: 458-471, 1980)을 일부 수정하여 사용하였다(Jeong, T. S., Bioorg. Med. Chem. Lett., 14: 2715-2717, 2004).Determination of ACAT activity was carried out using Brecher & P. (Brecher, P. and Co., Ltd.) using [1- 14 C] oleoyl-CoA (56.0 μCi / μmol; Amersham) as substrate. Chan, C., Biochim. Biophys. Acta, 617: 458-471, 1980) with some modifications (Jeong, TS, Bioorg. Med. Chem. Lett., 14: 2715-2717, 2004).
상기 실시예에서 제조한 초피 추출물 또는 그로부터 분리한 화합물 10 ㎕, 상기 실험예 2-1에서 제조한 hACAT-1 또는 hACAT-2 마이크로좀 용액 4.0 ㎕, 활성분석 완충액(0.5 M KH2PO4, 10 mM DTT, pH 7.4; Sigma, USA) 20.0 ㎕, 지방이 제거된 우혈청알부민(BSA; bovine serum albumin, 저장액 농도 40 ㎎/㎖; Sigma, USA) 15.0 ㎕, 콜레스테롤(저장액 농도 20 ㎎/㎖; Sigma, USA) 2.0 ㎕, 증류수 41.0 ㎕를 가하여 37℃에서 15 분간 예비 반응시켰다. 이 반응액에 [1-14C] 올레오일-코에 이(0.05 μCi, 최종농도: 50 μM) 8 ㎕를 첨가하여 다시 37℃에서 30 분간 반응시킨 후, 이소프로판올 : 헵탄 혼합물(4 : 1 (v/v)) 1 ㎖을 가하여 반응을 정지시켰다. 여기에 600 ㎕의 헵탄과 200 ㎕의 0.1 M KH2PO4(pH 7.4)을 첨가하고, 혼합물을 볼텍서(vortexer)로 격렬하게 혼합한 후, 300 × g에서 5 분 동안 원심분리를 하였다. 원심분리하여 얻은 100 ㎕의 상층액을 신틸레이션 병(scintillation bottle)에 넣고, 신틸레이션 액(Lipoluma) 4 ㎖을 가하였다. 이 혼합물의 방사선량은 1450 마이크로베타 액체 신틸레이션 계수기(1450 Microbeta liquid scintillation counter, Wallac Oy)로 측정하였다.10 μl of the extract of Chopi or the compound isolated therefrom, 4.0 μl of the hACAT-1 or hACAT-2 microsome solution prepared in Experimental Example 2-1, activity assay buffer (0.5 M KH 2 PO 4 , 10 20.0 μl mM DTT, pH 7.4; Sigma, USA), 15.0 μl fat-free bovine serum albumin (BSA; stock solution concentration 40 mg / ml; Sigma, USA), cholesterol (stock concentration 20 mg / Sigma, USA) 2.0 µl and distilled water 41.0 µl were added and preliminarily reacted at 37 ° C for 15 minutes. 8 µl of [1- 14 C] oleoyl-coe (0.05 µCi, final concentration: 50 µM) was added to the reaction mixture, and the resultant was reacted again at 37 ° C for 30 minutes, followed by isopropanol: heptane mixture (4: 1 ( 1 ml of v / v)) was added to stop the reaction. To this was added 600 μl heptane and 200 μl 0.1 M KH 2 PO 4 (pH 7.4), the mixture was vigorously mixed with a vortexer and centrifuged at 300 × g for 5 minutes. 100 μl of the supernatant obtained by centrifugation was placed in a scintillation bottle, and 4 ml of scintillation liquid (Lipoluma) was added thereto. The radiation dose of this mixture was measured with a 1450 Microbeta liquid scintillation counter (Wallac Oy).
ACAT 활성은 측정된 방사선량으로부터 시간당 방사선량을 계산하여 1 분 동안 단백질 1 ㎎당 합성된 콜레스테릴 올레이트 피코몰(피코몰/분/㎎ 단백질)로 계산하였다.ACAT activity was calculated from the synthesized cholesteryl oleate picolol (picomol / min / mg protein) per mg of protein for 1 minute by calculating the radiation dose per hour from the measured radiation dose.
결과는 표 1에 나타내었다. The results are shown in Table 1.
표 1에 나타난 바와 같이, 본 발명에 의한 초피 추출물, 그로부터 분리한 산스울계 화합물은 hACAT-1 및 hACAT-2에 대한 저해 활성이 우수함을 알 수 있었다. As shown in Table 1, the chopi extract according to the present invention, the acid-based compounds isolated therefrom were found to have excellent inhibitory activity against hACAT-1 and hACAT-2.
따라서 본 발명에 의한 초피 추출물, 그로부터 분리한 산스울계 화합물은 콜레스테릴 에스테르의 합성 및 축적으로 유발되는 고지혈증, 관상동맥 심장병, 동맥경화 및 심근경색증 등과 같은 심장순환계 질환의 예방 또는 치료용 조성물에 유용하게 사용할 수 있음을 확인하였다.Therefore, Chopi extract according to the present invention, the acid-based compounds isolated therefrom are useful for the prevention or treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction caused by the synthesis and accumulation of cholesteryl esters. It was confirmed that it can be used.
<실험예 2> 본 발명에 따른 초피 추출물 및 산스울계 화합물의 급성 독성실험Experimental Example 2 Acute Toxicity Test of Chopi Extract and Sansul Compounds According to the Present Invention
본 발명의 초피 추출물, 그로부터 분리한 산스울계 화합물에 대한 급성 독성을 알아보기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the acute toxicity of the chopi extract of the present invention, the acid-based compounds isolated therefrom, the following experiment was performed.
4주령의 특정 병원체 부재(SPF, specific pathogens free) ICR계 마우스를 암수 각각 3 마리씩 4개군(암수 각각 3마리/실험군)으로 나누어, 온도 22±3℃, 습도 55±10%, 조명 12L/12D 조건의 동물실 내에서 사육하였다. 마우스는 실험에 사용되기 전 1주일 정도 순화시켰다. 실험 동물용 사료(마우스 및 랫트용, Dyets, 미국) 및 음수를 멸균한 후 공급하였으며 자유 섭취시켰다.SPF (specific pathogens free) ICR mice were divided into 4 groups (3 females and 3 females / experimental group) at 4 weeks of age, temperature 22 ± 3 ° C, humidity 55 ± 10%, and illumination 12L / 12D. It was bred in a condition animal room. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (for mice and rats, Dyets, USA) and negative water were supplied after sterilization and free intake.
상기 실시예에서 제조한 초피나무 지상부 추출물, 초피 열매껍질 추출물, 그로부터 분리한 산스울계 화합물을 0.5% 트윈(tween) 80에 50 ㎎/㎖ 농도로 조제한 후, 마우스 체중 20 g 당 0.04 ㎖(100 ㎎/㎏), 0.2 ㎖(500 ㎎/㎏) 및 0.4 ㎖(1,000 ㎎/㎏)씩 경구 투여하였다. 시료는 단회 경구 투여하였으며, 투여 후 7일 동안 다음과 같이 부작용 또는 치사 여부를 관찰하였다. 즉, 투여 당일은 투여 후 1 시간, 4 시간, 8 시간, 12 시간 뒤에, 그리고 투여 익일부터 7 일째까지는 매일 오전, 오후 1 회 이상씩 일반 증상의 변화 및 사망 동물의 유무를 관찰하였다. 또한, 투여 7 일째에 동물을 치사시켜 해부한 후 육안으로 내부 장기를 검사하였다. 투여 당일부터 1일 간격으로 체중의 변화를 측정하여 초피나무 지상부 추출물, 초피 열매껍질 추출물, 그로부터 분리한 산스울계 화합물에 의한 동물의 체중 감소 현상을 관찰하였다.The ground extract of the ground bark, the bark fruit bark extract prepared in the above example, and the sansul-based compound separated therefrom were prepared at a concentration of 50 mg / ml in 0.5% tween 80, and then 0.04 ml (100 mg) per 20 g of mouse body weight. / Kg), 0.2 ml (500 mg / kg) and 0.4 ml (1,000 mg / kg). Samples were administered orally once and observed for side effects or lethality for 7 days after administration. That is, on the day of administration, changes in general symptoms and the presence or absence of dead animals were observed at least 1 hour, 4 hours, 8 hours, 12 hours after the administration, and at least once every morning and afternoon from the day after the administration. In addition, on the 7th day of administration, animals were killed and dissected, and the internal organs were visually examined. Changes in body weight were measured at intervals of 1 day from the day of administration, and the weight loss of the animals by the extract of ground bark of the bark, the bark of the bark fruit, and the acid-based compounds isolated therefrom were observed.
실험 결과, 시료를 투여한 모든 마우스에서 특기할 만한 임상증상이 나타나지 않았고 폐사된 마우스도 없었으며, 또한 체중변화, 혈액검사, 혈액생화학 검사, 부검소견 등에서도 독성변화는 관찰되지 않았다.As a result, no significant clinical symptoms were observed in all the mice treated with the sample, no mice died, and no toxicity change was observed in weight change, blood test, blood biochemistry test, autopsy findings, etc.
따라서, 본 발명에 따른 초피나무 지상부 추출물, 초피 열매껍질 추출물, 그로부터 분리한 산스울계 화합물은 모든 마우스에서 1,000 ㎎/㎏까지 독성변화를 나타내지 않았으며, 경구투여 최소치사량(LD50)이 1,000 ㎎/㎏ 이상인 안전한 물질로 판단되었다.Therefore, the ground extract of the ground bark of the present invention, the extract of the bark of the bark, and the extract from the Sanswoo-based compound did not show toxicity change up to 1,000 mg / kg in all mice, and the minimum lethal dose (LD 50 ) of 1,000 mg / It was judged to be a safe substance of more than kg.
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of preparations for the compositions of the present invention are illustrated below.
<제제예 1> 약학적 제제의 제조Preparation Example 1 Preparation of Pharmaceutical Formulation
<1-1> 산제의 제조<1-1> Preparation of Powder
본 발명에 따른 초피 추출물 또는 산스울계 화합물 2 gChopi extract or sansulul compound 2 g according to the present invention
유당 1 g1 g lactose
상기의 성분을 혼합한 후, 기밀포에 충진하여 산제를 제조하였다.After mixing the above components, the airtight cloth was filled to prepare a powder.
<1-2> 정제의 제조<1-2> Preparation of Tablet
본 발명에 따른 초피 추출물 또는 산스울계 화합물 100 ㎎100 mg of Chopi extract or Sansool compound according to the present invention
옥수수전분 100 ㎎Corn starch 100 mg
유 당 100 ㎎Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
<1-3> 캡슐제의 제조<1-3> Preparation of Capsule
본 발명에 따른 초피 추출물 또는 산스울계 화합물 100 ㎎100 mg of Chopi extract or Sansool compound according to the present invention
옥수수전분 100 ㎎Corn starch 100 mg
유 당 100 ㎎Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
<1-4> 주사액제의 제조<1-4> Preparation of Injection Solution
본 발명에 따른 초피 추출물 또는 산스울계 화합물 10 ㎍/㎖Chopi extract or Sanswooul compound 10 ㎍ / ㎖ according to the present invention
묽은 염산 BP pH 3.5로 될 때까지Dilute hydrochloric acid BP until pH 3.5
주사용 염화나트륨 BP 최대 1 ㎖Injectable sodium chloride BP up to 1 ml
적당한 용적의 주사용 염화나트륨 BP 중에 본 발명에 따른 초피 추출물, 산스울계 화합물을 용해시키고, 생성된 용액의 pH를 묽은 염산 BP를 사용하여 pH 3.5로 조절하고, 주사용 염화나트륨 BP를 사용하여 용적을 조절하고 충분히 혼합하였다. 용액을 투명 유리로 된 5 ㎖ 타입 I 앰플 중에 충전시키고, 유리를 용해시킴으로써 공기의 상부 격자하에 봉입시키고, 120℃에서 15 분 이상 오토클래이브시켜 살균하여 주사액제를 제조하였다.In the appropriate volume of sodium chloride BP for injection, disintegrate Chopi extract according to the present invention, the acid-based compound, adjust the pH of the resulting solution to pH 3.5 using dilute hydrochloric acid BP, and adjust the volume with the injection of sodium chloride BP. And mixed well. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.
<제제예 2> 식품의 제조Preparation Example 2 Preparation of Food
본 발명에 따른 초피 추출물 또는 산스울계 화합물을 포함하는 식품들을 다음과 같이 제조하였다.Foods containing chopi extract or Sansul-based compounds according to the present invention were prepared as follows.
<2-1> 밀가루 식품의 제조<2-1> Preparation of Flour Food
본 발명에 따른 초피 추출물 또는 산스울계 화합물 각각 0.1 ~ 10.0 중량부를 밀가루에 첨가하고, 이 혼합물을 이용하여 통상의 방법으로 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.0.1 to 10.0 parts by weight of each extract of Chopi extract or Sanswoo based compound according to the present invention was added to wheat flour, and bread, cake, cookies, crackers and noodles were prepared in a conventional manner using the mixture to prepare foods for health promotion.
<2-2> 스프 및 육즙(gravies)의 제조<2-2> Preparation of Soups and Gravys
본 발명에 따른 초피 추출물 또는 산스울계 화합물 각각 0.1 ~ 1.0 중량부를 스프 및 육즙에 첨가하여 통상의 방법으로 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.0.1 to 1.0 parts by weight of each extract of Chopi extract or Sanswoo based compound according to the present invention was added to soups and broths to prepare meat products for health promotion, soups and broths of noodles in a conventional manner.
<2-3> 그라운드 비프(ground beef)의 제조<2-3> Preparation of Ground Beef
본 발명에 따른 초피 추출물 또는 산스울계 화합물 각각 10 중량부를 그라운드 비프에 첨가하여 통상의 방법으로 건강 증진용 그라운드 비프를 제조하였다.The ground beef for health promotion was prepared by adding 10 parts by weight to each ground beef of chopi extract or sanswool compound according to the present invention.
<2-4> 유제품(dairy products)의 제조<2-4> Production of Dairy Products
본 발명에 따른 초피 추출물 또는 산스울계 화합물 각각 0.1 ~ 1.0 중량부를 우유에 첨가하고, 상기 우유를 이용하여 통상의 방법으로 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.0.1 to 1.0 parts by weight of each of the extract of Chopi or sansool-based compound according to the present invention was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.
<2-5> 선식의 제조<2-5> Preparation of Wire
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.
검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.
본 발명에 따른 초피 추출물 또는 산스울계 화합물을 진공 농축기에서 감압, 농축하고, 분무, 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60 메쉬로 분쇄하여 건조분말을 얻었다.Chopi extract or sanswooul compound according to the present invention was decompressed, concentrated in a vacuum concentrator, dried by spraying, drying with a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.
상기에서 제조한 곡물류, 종실류 및 본 발명에 따른 초피 추출물 또는 산스울계 화합물의 건조분말을 다음의 비율로 배합하여 통상의 방법으로 제조하였다.The grains, seeds, and dried powders of the extract of Chopi or sansul-based compounds according to the present invention were prepared in the following ratio to prepare a conventional method.
곡물류(현미 30 중량부, 율무 15 중량부, 보리 20 중량부),Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),
종실류(들깨 7 중량부, 검정콩 8 중량부, 검정깨 7 중량부),Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),
본 발명에 따른 초피 추출물 또는 산스울계 화합물의 건조분말(1 중량부),Dry powder (1 part by weight) of the extract of Chopi or sanswool compound according to the present invention,
영지(0.5 중량부),Ganoderma lucidum (0.5 parts by weight),
지황(0.5 중량부)Foxglove (0.5 part by weight)
<제제예 3> 음료의 제조Preparation Example 3 Preparation of Beverage
본 발명에 따른 초피 추출물 또는 산스울계 화합물을 포함하는 음료를 다음과 같이 제조하였다.The beverage containing the extract of Chopi or sansool-based compound according to the present invention was prepared as follows.
<3-1> 건강음료의 제조<3-1> Preparation of health drink
액상과당(0.5%), 올리고당(2%), 설탕(2%), 식염(0.5%), 물(75%)과 같은 부재료와 본 발명에 따른 초피 추출물 또는 산스울계 화합물을 균질하게 배합하여 순간 살균을 한 후, 이를 유리병, 패트병 등 소포장 용기에 포장하여 건강음료를 제조하였다.Instantly mix homogeneous ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%), water (75%) and Chopi extract or Sanswool compound according to the present invention. After sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare healthy drinks.
<3-2> 야채쥬스의 제조<3-2> Preparation of Vegetable Juice
본 발명에 따른 초피 추출물 또는 산스울계 화합물 0.5 g을 토마토 또는 당근 등의 야채의 쥬스 1,000 ㎖에 가하여 통상의 방법으로 건강 증진용 야채쥬스를 제조하였다.0.5 g of the chopi extract or the acid-based compound according to the present invention was added to 1,000 ml of vegetable juice, such as tomato or carrot, to prepare vegetable juice for health promotion in a conventional manner.
<3-3> 과일쥬스의 제조<3-3> Preparation of fruit juice
본 발명에 따른 초피 추출물 또는 산스울계 화합물 0.1 g을 사과 또는 포도 등의 과일의 쥬스 1,000 ㎖에 가하여 통상의 방법으로 건강 증진용 과일쥬스를 제조하였다.0.1 g of Chopi extract or Sansool compound according to the present invention was added to 1,000 ml of fruit such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.
본 발명의 초피 추출물 또는 그로부터 분리된 산스울계 화합물은 아실-코에이:콜레스테롤 전이효소(acyl-CoA:cholesterol acyltransferase; ACAT)의 활성을 효과적으로 억제할 뿐 아니라 마우스에서 급성독성을 나타내지 않는다.Chopi extract of the present invention or the acid-based compounds isolated therefrom not only effectively inhibit the activity of acyl-CoA: cholesterol acyltransferase (ACAT) but also do not exhibit acute toxicity in mice.
따라서, 본 발명의 조성물은 콜레스테릴 에스테르의 합성 및 축적으로 유발되는 고지혈증, 관상동맥 심장병, 동맥경화 및 심근경색증 등과 같은 심장순환계 질환의 예방 및 치료용 조성물에 유용하게 사용될 수 있다.Therefore, the composition of the present invention can be usefully used in the composition for the prevention and treatment of cardiovascular diseases such as hyperlipidemia, coronary heart disease, arteriosclerosis and myocardial infarction caused by the synthesis and accumulation of cholesteryl ester.
Claims (13)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020060109551A KR100883992B1 (en) | 2006-11-07 | 2006-11-07 | Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020060109551A KR100883992B1 (en) | 2006-11-07 | 2006-11-07 | Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases |
Publications (2)
Publication Number | Publication Date |
---|---|
KR20080041441A KR20080041441A (en) | 2008-05-13 |
KR100883992B1 true KR100883992B1 (en) | 2009-02-17 |
Family
ID=39648608
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
KR1020060109551A KR100883992B1 (en) | 2006-11-07 | 2006-11-07 | Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases |
Country Status (1)
Country | Link |
---|---|
KR (1) | KR100883992B1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021230667A1 (en) | 2020-05-13 | 2021-11-18 | 메콕스큐어메드 주식회사 | Coronavirus therapeutic agent comprising zanthoxylum piperitum leaf extract as active ingredient |
KR20210139180A (en) | 2020-05-13 | 2021-11-22 | 메콕스큐어메드 주식회사 | Therapeutic agent for coronavirus comprising Zanthoxylum piperitum leaf extract as effective component |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102143968B1 (en) * | 2018-10-15 | 2020-08-12 | 한국과학기술연구원 | Compositions for for inhibiting of macrophage death by triglycerides comprising extracts of Angelica takeshimana |
CN114835582B (en) * | 2022-04-29 | 2024-03-15 | 贵州黄果树立爽药业有限公司 | Recycling method of Liangfu drop pill extract solvent |
CN115304508B (en) * | 2022-08-08 | 2023-07-25 | 四川大学华西医院 | Bionic antibacterial and antioxidant nanoparticle based on pricklyash peel extract, and preparation method and application thereof |
CN116196415B (en) * | 2022-12-07 | 2023-08-01 | 浙江省肿瘤医院 | Mixed formulations for sensitizing PD-1 antibodies and methods of use thereof |
-
2006
- 2006-11-07 KR KR1020060109551A patent/KR100883992B1/en not_active IP Right Cessation
Non-Patent Citations (4)
Title |
---|
Phytochemistry 21(6), 1295-8 (1982) * |
Phytochemistry 21(6), 1295-8 (1982)* |
한국식품영양과학회지 35(1), 21-27 (2006. 01.) * |
한국식품영양과학회지 35(1), 21-27 (2006. 01.)* |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2021230667A1 (en) | 2020-05-13 | 2021-11-18 | 메콕스큐어메드 주식회사 | Coronavirus therapeutic agent comprising zanthoxylum piperitum leaf extract as active ingredient |
KR20210139180A (en) | 2020-05-13 | 2021-11-22 | 메콕스큐어메드 주식회사 | Therapeutic agent for coronavirus comprising Zanthoxylum piperitum leaf extract as effective component |
Also Published As
Publication number | Publication date |
---|---|
KR20080041441A (en) | 2008-05-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
KR100883992B1 (en) | Composition containing extracts of Zanthoxylum piperitum DC or compounds isolated therefrom for the prevention and treatment of cardiovascular diseases | |
KR100704299B1 (en) | Novel quinoline compound, and composition containing centipede extract or compounds isolated therefrom for prevention and treatment of cardiovascular disease | |
US7825162B2 (en) | Abietane diterpenoid compound, and composition comprising extract of torreya nucifera, or abietane diterpenoid compounds or terpenoid compounds isolated from them for prevention and treatment of cardiovascular disease | |
KR20090010504A (en) | Compositions for the prevention and treatment of cardiovascular diseases containing sophora flavascens extracts, soluble extracts thereof, fractions thereof or flavonoid compounds isolated therefrom as an active ingredient | |
KR100823155B1 (en) | Novel catecholic xanthone compounds, and composition containing extract of Cudrania tricuspidata or catecholic xanthone compounds isolated therefrom for the prevention and treatment of cardiovascular disease | |
KR100772495B1 (en) | Composition comprising extract of Torreya nucifera or abietane diterpenoid compounds isolated from them for prevention and treatment of cardiovascular disease | |
KR100836189B1 (en) | Composition containing extracts of Houttuynia cordata Thunb. or lignan compounds, dihydroguaiaretic acid, isolated from there for the prevention and treatment of cardiovascular disease | |
KR100778373B1 (en) | therapeutic agent comprising Mantidis ootheca extracts, N-acetyldopamine or biphenol compounds isolated from the same for prevention and treatment of cardiovascular disease | |
JP2010270096A (en) | Glucose absorption inhibitor | |
KR100559495B1 (en) | Composition comprising dineolignan compounds for inhibiting acyl-CoA:cholesterol acyltransferase | |
KR100703859B1 (en) | Composition comprising extract of Lycopus lucidus Turcz. for the prevention and treatment of cardiovascular disease | |
KR100590726B1 (en) | Composition comprising extract of Phellinus sp. PL3 or Phellinsin A isolated from the same as an effective component for prevention and treatment of cardiac circuit disease | |
KR20190124194A (en) | Barley sprout tea having increased content of antioxidative or hypoglycemic components | |
KR100575253B1 (en) | Novel abietane diterpenoid compounds for prevention and treatment of cardiovascular disease and the composition comprising the same | |
KR100629314B1 (en) | Composition comprising extract of Alnus japonica Steud. or diarylheptanoid compounds isolated from them for prevention and treatment of cardiovascular disease | |
KR100758263B1 (en) | New n-acetyldopamine compounds, and the composition comprising periostracum cicadae extract or n-acetyldopamine compounds isolated from the same for prevention and treatment of cardiovascular disease | |
KR100542871B1 (en) | Composition comprising dineolignan compounds for inhibiting acyl-CoA:cholesterol acyltransferase | |
KR100631486B1 (en) | Preventive and therapeutic agent for cardiopulmonary diseases with terpenoid compounds as an active ingredient | |
KR100806226B1 (en) | A composition for the prevention and treatment of cardiovascular disease containing extract of Glycine max Roots or polypenol compounds isolated thereof | |
KR100588358B1 (en) | Agent comprising terpenoid compounds for prevention and treatment of cardiovascular disease | |
KR20130112980A (en) | Composition comprising cleistocalyx operculatus extract and compound isolated from the same for preventing or treating atherosclerosis | |
KR102428082B1 (en) | Cosmetic composition, pharmaceutical composition, and food composition comprising pterocaria stenoptera extract, which have anti-inflammatory immune activity and antioxidant activity and provide excellent effect on muscle cell generation for strengthening muscle strength or muscle in the elderly | |
KR100724088B1 (en) | Composition containing flavonoid compounds for the prevention and treatment of cardiovascular disease | |
KR20170076587A (en) | Composition comprising Monoterpenyl magnolol as an effective ingredient for preventing or treating of obesity, hyperlipidemia or fatty Liver and Method for preparing fraction of Magnolia cortex | |
KR100778031B1 (en) | A composition comprising glycerol compounds for prevention and treatment of cardiovascular disease |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
A201 | Request for examination | ||
E902 | Notification of reason for refusal | ||
E902 | Notification of reason for refusal | ||
E90F | Notification of reason for final refusal | ||
E701 | Decision to grant or registration of patent right | ||
GRNT | Written decision to grant | ||
FPAY | Annual fee payment |
Payment date: 20130123 Year of fee payment: 5 |
|
FPAY | Annual fee payment |
Payment date: 20140211 Year of fee payment: 6 |
|
FPAY | Annual fee payment |
Payment date: 20160121 Year of fee payment: 8 |
|
LAPS | Lapse due to unpaid annual fee |